TWO-PHOTON SINGLE PARTICLE TRACKING IN THREE DIMENSIONS

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An understanding of the kinetics of transport processes is crucial in biology and medicine. On the cellular level, examples of important transport processes include endocytosis of extracellular protein, organelle transport during mitosis, phagocytosis of antigen material, mRNA nucleo-cytoplasmic transport, virus docking and infection. Trafficking inside a complex three-dimension environment is a shared common theme. These trafficking processes are rarely passive or diffusion-controlled in cellular system but are guided through active mechanisms including molecular motors and ion pumps. These complex sequences of events are difficult to resolve when the action of many cells are asynchronously averages. Singe particle tracking (SPT) was developed in the early 1980's to address this problem and has proved to be a powerful technique to further our understanding of membrane protein diffusion, membrane compartmentalization and protein-cytoskeleton interaction. However, the traditional SPT method which uses wide field to the study of two-dimensional systems. In this proposal, the applicant plans to develop a 3-D particle tracking technique utilizing the inherent sub-femtoliter localization of two photon excitation. Using a real-time feedback system, he can dynamically position this excitation volume to follow the position of a fluorescent particle under transport by maximizing detected fluorescent intensity. It has been well established that the sequence of cellular transport are often controlled by biochemical signals. Fluorescent spectroscopic method applied in microscopy setting can provide sensitive and specific sampling of the local cellular biochemical states. Dr. So proposes to integrate wavelength resolved spectroscopy with 3D-SPT to continuously monitor the particle micro- environment along its transpiration path. The first application of this instrument will be focused on the study of receptor-mediate protein endocytosis.